1. Field of the Invention
The invention relates to a flask used in the culturing of cells or tissue. The flask may be used in the preparation of cultured cells or of conditioned media from the cells. More particularly, a flask and method providing for continuous dialysis of the culture media is disclosed. The flask and method are particularly useful for the preparation of conditioned media from tissues or various cell populations or for the long term in vitro growth and maintenance of cells.
2. Description of the Prior Art
The need exists for methods of preparing quantities of conditioned media from tissues or various cell populations. The preparation of conditioned media is a routine procedure in tissue culture and hemopoiesis laboratories which are involved in investigating various cell-cell interactions and other regulatory phenomena. Long term in vitro growth and maintenance of cells is also of interest in these laboratories.
The prior art preparation of conditioned media is a multi-step process. First, the conditioned media is prepared in a Petri dish or a simple tissue culture flask, such as a flat stoppered tissue culture flask. After preparation, the media is harvested and dialyzed. The dialysis step removes putative low molecular weight inhibitors of cell growth. This multi-step process involves considerable manipulation of the conditioned media, resulting in the loss of some quantities of the media. This loss may be significant when rather small quantities of conditioned media are prepared. More importantly, this manipulation may result in some heretofore unanticipated inadvertent changes in the qualities of the conditioned media.
The prior art methods of growing long term in vitro cell cultures generally include the use of flat tissue culture flasks. Cells are introduced into the flasks in a suspension in the culture media supplemented with horse serum and other nutrients. The flat tissue culture flasks are then incubated lying on their flat sides. A part of the culture media is withdrawn and replaced with fresh media at frequent intervals. Often half of the media is replaced twice a week. This preparation method is known as the "Dexter Method". See Dexter, T. M., "Cell Interactions in vitro", Clinics in Haematology 8, 453-468 (1979), at 454-455 for a specific example of the "Dexter Method." Although the "Dexter Method" is able to maintain mouse haemopoietic stem cells for as many as twelve to sixteen weeks, it is unable to maintain human stem cells beyond a period of four to five weeks.
Disadvantages of the "Dexter Method" include the accumulation of putative inhibitory molecules in the culture media necessitating frequent replacement of a portion of the culture media. Another disadvantage associated with the frequent replacement of the culture media is the removal of stimulatory macromolecules essential for the maintenance and growth of the required cells. These factors may be responsible for the inability to maintain long term growth of the cultures.
A prior art method providing for continuous dialysis is the "Marbrook System" and its modifications. See Marbrook, J., "Primary Immune Response in Cultures of Spleen Cells," The Lancet 2, 1279-1281 (1967), at 1280; Feldmann, M., and A. Basten, "Cell Interactions in the Immune Response in Vitro III. Specific Collaboration Across a Cell Impermeable Membrane," J. Exp. Med. 136, 49-67 (1972), at 51. The Marbrook System is often used for investigating the effects of humoral factors elaborated by one cell population over another. The Marbrook System employs a flask similar to an Erlenmeyer flask to contain the dialysate solution. An inner chamber with a dialysis or millipore membrane sealed to the open end contains the culture media and cells. This inner chamber is partially submerged in the dialysis solution of the outer chamber. The cells to be cultured are placed directly on the dialysis membrane.
Disadvantages of the "Marbrook System" include the inability to maintain and establish long-term tissue cultures. Other disadvantages are the problems associated with the clogging of the dialysis membrane caused by placing the cells to be cultured directly on the membrane. Still other problems are associated with the microenvironment on the dialysis membrane.
Accordingly, prior to the development of the present invention, there has been no cell culture and continuous dialysis flask and method which solves the problems and overcomes the disadvantages associated with the prior art flasks and methods discussed above. The flask and method described by the present invention is able to culture cells or tissue over long periods of time while simultaneously dialyzing the conditioned culture media under preparation. This results in a significant saving of time and in a minimization of manipulation of the media. This minimization of manipulation results in less contamination, change and loss of the media. The flask and method described by the present invention does not require frequent replacement of the culture media. Thus putative inhibitory molecules in the culture media are removed by continuous simultaneous dialysis while stimulatory macromolecules remain unaffected. The flask and method described by the present invention provides a tissue culture shelf for the establishment of a "feeder layer" of the cells to be cultured. This shelf helps to prevent the clogging of the dialysis membrane and the problems associated therewith.
The art has long sought a tissue culture flask and method which could be used to prepare conditioned media or to grow cells for long terms in vitro by providing continuous simultaneous dialysis of the culture media. As shown below, the flask and method of the present invention meets this need.